Recombinant proteins have many uses in biotechnology whenever large amounts of pure protein are needed. Microbial expression systems such as Escherichia coli (E. coli) and yeast are often the first choice due to their low cost and high yield. When expressing foreign proteins in E. coli, it is not uncommon to encounter problems of low levels of expression and/or insolubility of the protein. Even if the protein is expressed well and remains soluble, it must be purified from the myriad of other proteins in the cell extract. To facilitate the expression and purification of a target protein, one method that is in common use is to fuse an affinity tag to the protein. A good affinity tag has properties that facilitate high-level expression when fused to the N-terminus of the target protein, and provides a simple one-step affinity purification that allows the target protein to be purified from the expression milieu.
The maltose-binding protein (MBP) of E. coli is commonly used as an affinity tag for expression and purification of foreign proteins produced in E. coli. The natural role of MBP is to bind maltodextrins at the outer membrane porin and release them to the MalEFK transport apparatus in the inner membrane. Fusion of the C-terminus of MBP to the N-terminus of a target protein permits the expression of the fusion protein in E. coli (FIG. 1). MBP and MBP fusions can be purified in a single step by binding to a chromatography matrix containing any of a number of glucose-α1→4-glucose polysaccharides such as amylose, starch or other maltodextrins (U.S. Pat. No. 5,643,758). Many proteins that are soluble in their native host are insoluble when expressed as a recombinant protein. For many of these proteins, fusion to MBP renders them soluble (Kapust & Waugh, Protein Sci. 8:1668-74 (1999)).
The utility of MBP as an affinity tag is tempered by the fact that depending on the protein in a MBP-target protein purification, some fusions don't bind to the affinity matrix as well as others. In addition, the presence of non-ionic detergents such as Triton X100 and Tween 20 can interfere with binding, especially for MBP-target protein fusions that have an inherently lower affinity.
Researchers have used the structure of MBP to make directed mutations in order to alter the binding properties of MBP. The X-ray crystal structure of MBP has been reported by Spurlino et al., J. Biol. Chem. 266:5202-5219 (1991). MBP consists of two domains, with a cleft between the domains where the polysaccharide binds. The domain that contains the N-terminus is named the domain I, and the domain that contains the C-terminus is named the domain II. Three loops cross between the two domains to form a hinge. Two groups have used the structure to make directed mutations to the region behind the hinges that increase the affinity of MBP for maltose and maltotriose (Marvin et al., Nature Structural Biology 8:795-798 (2001); Telmer & Shilton, Journal of Biol. Chem. 278:34555-34567 (2003)). However, this approach has an inherent disadvantage, since these modifications to MBP increase the hydrophobicity of the surface of the protein and thus decrease its solubility. This reduces its utility as an affinity tag by increasing its tendency to render a fusion protein insoluble.